The protein phosphatases are intrinsic components of the regulatory mechanisms involving protein phosphorylation-dephosphorylation. The aims of the research are the rigorous isolation and characterization of two major types of protein phosphatases in rabbit skeletal muscle, focusing on the determination of their molecular properties, potential interrelationships and regulation. A better knowledge of these enzymes may contribute to our understanding of a major regulatory mechanism which involves both carbohydrate metabolism and muscle contractility. This basic knowledge may expand our understanding of diseases involving either carbohydrate or muscle dysfunction, including diabetes. The specific aims are as follows: Characterization of protein phosphatases C-I and C-II. These are two Mr = ca. 35,000 protein phosphatases which have been isolated as the putative catalytic subunits of larger holoenzymes. Their potential relationships will be investigated by structural analysis of the two proteins and immunochemical means employing monoclonal antibodies. Isolation and characterization of the high molecular weight protein phosphatases. Phosphatases H-I and H-II will be purified to homogeneity and their properties and subunit structures determined. The nature of their catalytic subunits will be studied and compared to phosphatase C-I and C-II. The phenomenon of the in vitro activation of purified phosphatases C-II and H-II by insulin will be further investigated. Monoclonal antibodies to the protein phosphatases will be isolated. Monoclonal antibodies toward the four protein phosphatases listed above will be isolated. These antibodies will be characterized and used to study the immunochemical relationships between the protein phosphatases, for immunoaffinity purification of the phosphatases, for immunolocalization studies, and as probes for the topographical relationships of antigenic determinants. Use of the skinned muscle fiber system to study the functional specificities of the protein phosphatases. The specificity and regulation of the protein phosphatases will be studied using a skinned muscle fiber preparation. The dephosphorylation of phosphorylase, glycogen synthase, the Alpha- and Beta-subunits of phosphorylase kinase and mysoin light chains in permeabilized muscle fibers by exogenous phosphatases, phosphatase inhibitors and monoclonal antibodies to the phosphatases will be studied.